Difference between revisions of "Marine mammals' health as an indicator of ecosystem health - tools for monitoring"

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{{Review
 
|name=James Everts
 
|AuthorID=26539
 
}}
 
  
  
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==Introduction==
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[[Image:Kakuschke_1.jpg|300px|thumb|left|'''Figure 1''': The health of the Harbour Seal as top predator is an important biological parameter of the Trilateral Monitoring and Assessment Program (TMAP). This program was founded by The Netherlands, Denmark and Germany, for the protection and conservation of the Wadden Sea. It includes management, monitoring and research, as well as political matters.]]
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[[Image:Kakuschke_2.jpg|350px|thumb|right|'''Figure 2''':Overview of immunological investigations using blood samples of marine mammals.]]
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Marine mammals are top predators of the marine [[food web]] and can therefore be used as indicators for the ecosystem (Trilateral Monitoring and Assessment Program, TMAP, Fig. 1). Industrial human activities, e.g. fisheries, offshore drilling and wind power generation, as well as [[pollutants]] in the environment affect the [[North Sea|North]] and [[Baltic Sea]] [[Ecosystem|ecosystems]], which are the [[habitat]] of marine mammals such as harbour porpoises (''[http://www.marinespecies.org/aphia.php?p=taxdetails&id=137117 Phocoena phocoena]''), harbour seals (''[http://www.marinespecies.org/aphia.php?p=taxdetails&id=137084 Phoca vitulina]'') and grey seals (''[http://www.marinespecies.org/aphia.php?p=taxdetails&id=137080 Halichoerus grypus]'').
 +
The present article reports tools to investigate the health status of harbour seals. A dysregulation of the immune system can lead either to suppression or an increased sensitivity (hypersensitivity) of the immune system, which might affect morbidity and mortality in these animals. Parameters of the immune system were investigated as stable biomarkers for disease processes. Furthermore, factors indicating the impact of pollutants were identified and chemically characterised.
  
===Introduction===
+
==Methodology==
[[Image:Kakuschke_2.jpg|300px|thumb|right|'''Figure 2''':Overview of immunological investigations using blood samples of marine mammals.]]Marine mammals, are used as indicators of ecosystem change (Trilateral Monitoring and Assessment Program, TMAP, Fig. 1). They are top predators in the marine food web. Increasing industrial and commercial activity, e.g. fisheries and offshore drilling and wind parks, as well as the input of pollutants, affect the North Sea and Baltic Sea ecosystems, including native marine mammals such as Harbour Porpoises (''Phocoena phocoena''), Harbour Seals (''Phoca vitulina'') and Grey Seals (''Halichoerus grypus''). [[Image:Kakuschke_1.jpg|300px|thumb|left|'''Figure 1''': The health of the Harbour Seal as top predator is an important biological parameter of the Trilateral Monitoring and Assessment Program (TMAP). This program was founded by The Netherlands, Denmark and Germany, for the protection and conservation of the Wadden Sea. It includes management, monitoring and research, as well as political matters.]]This article presents some tools for early diagnosis of the health status of Harbour Seals. The selected biomarkers are non-destructive and are parameters for the immune system, which plays a central role in the control of disease processes. The dysregulation of the immune system may lead to immune suppression or enhancement (hypersensitivity). The indicators for the effects of pollutants are identified and chemically characterised.
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The following parameters of the immune system can be measured in blood samples of captive and free-ranging animals using in-vivo measurements, in-vitro cell culture experiments, and bioanalytical methods (Fig. 2): lymphocyte proliferation, mRNA and protein expression of cytokines and acute phase proteins (APP). Protein structures, and the influence of metal and organic pollutants on these can be analysed and allow, therefore, conclusions about the relevance of the pollutant.  
 
+
Elements indicating the relationship between pollutant burden and health parameters can be analysed in body fluids using mass spectrometry (see article [[Elemental mass spectrometry - a tool for monitoring trace element contaminants in the marine environment|Elemental mass spectrometry]]).
===Methodology===
 
The following immune system parameters can be measured in blood samples of captive and wild living animals using biomolecular and biochemical methods(Fig. 2, Fig. 3): lymphocyte proliferation as important immune cell function, and the expression of cytokines released by immune cells, which participate in an immune reaction.
 
 
[[Image:Kakuschke_3.jpg|300px|thumb|left|'''Figure 3''': Directly after arrival in the Seal Station the lymphocytes of newborns were particularly susceptible to the toxic effect of metals. A lot of metals tested e.g. beryllium, lead and cadmium inhibit the lymphocyte proliferation (value <0.1). This effect decreased during the time of rehabilitation.]]
 
[[Image:Kakuschke_3.jpg|300px|thumb|left|'''Figure 3''': Directly after arrival in the Seal Station the lymphocytes of newborns were particularly susceptible to the toxic effect of metals. A lot of metals tested e.g. beryllium, lead and cadmium inhibit the lymphocyte proliferation (value <0.1). This effect decreased during the time of rehabilitation.]]
  
===Technique for measurement of lymphocyte proliferation===
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===Measurement of lymphocyte proliferation===
Lymphocytes are isolated from the blood sample and cultured with and without stimulation using a lymphocyte transformation test (LTT, Fig. 2). After incubation, transformation and proliferation are examined and a stimulation index is calculated.
+
Lymphocytes are isolated from blood samples and cultured with and without ions of metal pollutants using a lymphocyte transformation test (LTT, Fig. 2). After an incubation period, transformation and proliferation of the lymphocytes is examined and a stimulation index calculated. Depending on degree of stimulation the reactivity of the lymphocytes and therefore immune system can be detected.
  
===Technique for quantification of cytokine expression===
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===Quantification of cytokine expression===
Cytokine expression is measured by analysing the amount of mRNA with the real time reverse transcriptase polymerase chain reaction (RT-PCR, Fig. 2). Detecting the amount of the mRNA allows us to calculate ratios between cytokines and thus establish the main focus of the immune response. Investigations of the cytokine expression pattern (Interleukin-1, -2, -4, -6, -10, -12, TNF, TGFß) allow the status of the immune reaction to be differentiated, whether the emphasis is on the cellular or humoral (body liquid) immune response.
+
mRNA and protein expression of cytokines and APP can be measured by real time reverse transcriptase polymerase chain reaction (RT-PCR) and mass spectrometry, respectively. Next to quantification of protein concentrations, mass spectrometry allows also the characterization of protein isoforms (Fig. 2).  
 +
Cytokines as interleukin-1, -2, -4, -6, -10, -12, tumour necrosis factor-alpha, transforming growth factor-beta and APP (e.g. haptoglobin, C-reactive protein, transferrin, [[metallothionein]], heat shock protein) are stimulated by infection, inflammation, trauma or intoxication. They might therefore be helpful to identify an activated immune system and to monitor the health status.
  
 
[[Image:Kakuschke_4.jpg|thumb|300px|right|'''Figure 4''': Cytokine index of IL-2 mRNA from the blood samples of two harbour porpoises living in captivity (Pp1, Pp2) and four accidentally caught animals (Pp3-Pp6)(Fonfara et al. 2007)]]
 
[[Image:Kakuschke_4.jpg|thumb|300px|right|'''Figure 4''': Cytokine index of IL-2 mRNA from the blood samples of two harbour porpoises living in captivity (Pp1, Pp2) and four accidentally caught animals (Pp3-Pp6)(Fonfara et al. 2007)]]
  
===Metal pollution – effects on immune system===
+
==Metal pollution – effects on the immune system==  
Pollution by metals may affect the immuno-competence of free-ranging populations of marine mammals in many areas of the industrialised world. An imbalance of the immune system caused by pollutants has been suggested to play a role in the incidence of infectious diseases in marine mammals (Jepson et al., 1999<ref name="J">Jepson, P. D., Bennett, P. M., Allchin, C. R., Law, R. J., Kuiken, T., Baker, J. R., Rogan, E. & Kirkwood, J. K. (1999). Investigating potential associations between chronic exposure to polychlorinated biphenyls and infectious disease mortality in harbour porpoises from England and Wales. Science of the Total Environment, 244, 339-348.</ref>; Siebert et al., 1999<ref name="S">Siebert, U., Joiris, C., Holsbeek, L., Benke, H., Failing, K., Frese, K. & Petzinger, E. (1999). Potential relation between mercury concentrations and necropsy findings in cetaceans from German waters of the North and Baltic Seas. Marine Pollution Bulletin, 38 (4), 285-295.</ref>; Bennett et al., 2001<ref name="B">Bennett, P. M., Jepson, P. D., Law, R. J., Jones, B. R., Kuiken, T., Baker, J. R., Rogan, E. & Kirkwood, J. K. (2001). Exposure to heavy metals and infectious disease mortality in harbour porpoises from England and Wales. Environmental Pollution, 112 (1), 33-40.</ref>). Metals affect the function of immuno-competent cells by a variety of mechanisms. Depending on the particular metal, its speciation, concentration and bioavailability, and a number of other factors, a continuous metal exposure will result in immuno-suppression or immuno-stimulating effects.
+
 
 +
In many areas of the industrialised world pollution by metals is present, which might have an effect on the immune competence of free-ranging [[Pollution and marine mammals|marine mammal populations]]. An imbalance of the immune system caused by pollutants has been suggested to play a role in the incidence of infectious diseases in marine mammals (Jepson ''et al''., 1999<ref name="J">Jepson, P. D., Bennett, P. M., Allchin, C. R., Law, R. J., Kuiken, T., Baker, J. R., Rogan, E. & Kirkwood, J. K. (1999). Investigating potential associations between chronic exposure to polychlorinated biphenyls and infectious disease mortality in harbour porpoises from England and Wales. Science of the Total Environment, 244, 339-348.</ref>; Bennett ''et al''., 2001<ref name="B">Bennett, P. M., Jepson, P. D., Law, R. J., Jones, B. R., Kuiken, T., Baker, J. R., Rogan, E. & Kirkwood, J. K. (2001). Exposure to heavy metals and infectious disease mortality in harbour porpoises from England and Wales. Environmental Pollution, 112 (1), 33-40.</ref>). Metals affect the function of immune cells by a variety of mechanisms. Depending on the particular metal, its speciation, concentration and bioavailability, permanent metal exposure can result in suppression or stimulation of the immune system.
 +
 
 +
=== Metal induced hypersensitivity in seals===
  
==== - Metal induced hypersensitivity in seals====
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A chronic exposure to [[Heavy metals|metal]] pollutants and the intake via food and water was suspected to cause hypersensitivity reactions in marine mammals. Hypersensitivity reactions were found for Mo > Ni >Ti > Cr, Al > Pb, Be, Sn, (in decreasing frequency as indicated by the present order) in several pinnipeds from the North Sea (Kakuschke, 2006<ref name="K1">Kakuschke, A. (2006). Einfluss von Metallen auf das Immunsystem von Meeressäugern. Dissertation, Universität Hamburg.</ref>) and a relationship between blood concentrations and hypersensitivity reactions of the particular metals was present (Kakuschke ''et al''., 2005 <ref name="K2">Kakuschke, A., Valentine-Thon, E., Griesel, S., Fonfara, S., Siebert, U. & Prange, A. (2005). The immunological impact of metals in Harbor Seals (Phoca vitulina) of the North Sea. Environmental Science & Technology, 39 (19), 7568-7575.</ref>). Furthermore, an association between lymphocyte proliferation caused by Ni and Be and cytokine expression was detected in a study of a grey seal (Kakuschke ''et al''., 2006<ref name="K3">Kakuschke, A., Valentine-Thon, E., Fonfara, S., Griesel, S., Siebert, U. & Prange, A. (2006). Metal sensitivity of marine mammals: a case study of a gray seal. Marine Mammal Science, 22 (4), 985-997.</ref>). Changes in haematological parameters and liver enzymes in seals with a metal-induced hypersensitivity suggests that hypersensitivity reactions are a complex phenomenon (Kakuschke ''et al''., 2011 <ref name="k4"> Kakuschke, A., Valentine-Thon, E., Griesel, S., Fonfara, S., Siebert, U. & Prange, A. (2011). Are metal-induced hypersensitivities in harbor seals associated with liver function? Marine Pollution Bulletin, 62, 1891-1894.</ref>). These findings indicate a chronic exposure of marine mammals of the North Sea to metal pollutants.  
The chronic intake of metal pollutants makes marine mammals susceptible to developing hypersensitivity reactions. Metal-specific hypersensitivity reactions were found in different pinnipeds from the North Sea (Kakuschke, 2006<ref name="K1">Kakuschke, A. (2006). Einfluss von Metallen auf das Immunsystem von Meeressäugern. Dissertation, Universität Hamburg.</ref>). The frequency of sensitising metals was in the order Mo > Ni >Ti > Cr, Al > Pb, Be, Sn. A relationship was found between the blood levels of metals to metalspecific hypersensitivity reactions (Kakuschke et al., 2005<ref name="K2">Kakuschke, A., Valentine-Thon, E., Griesel, S., Fonfara, S., Siebert, U. & Prange, A. (2005). The immunological impact of metals in Harbor Seals (Phoca vitulina) of the North Sea. Environmental Science & Technology, 39 (19), 7568-7575.</ref>). A relationship between lymphocyte proliferation and cytokine expression could be shown: in a study of a grey seal, a hypersensitivity reaction to Ni and Be has been correlated to alterations in the cytokine pattern (Kakuschke et al., 2006<ref name="K3">Kakuschke, A., Valentine-Thon, E., Fonfara, S., Griesel, S., Siebert, U. & Prange, A. (2006). Metal sensitivity of marine mammals: a case study of a gray seal. Marine Mammal Science, 22 (4), 985-997.</ref>).
 
  
==== - High susceptibility of the immune system of pups to the toxic effect of metals====
 
Pups are exposed to metals due to the transplacental transfer mother/fetus, the transfer through the milk and later by contaminated prey. Kakuschke et al. (2007<ref name="K4">Kakuschke, A., Valentine-Thon, E., Fonfara, S., Griesel, S., Siebert, U. & Prange, A. (2008). Metal-Induced Impairment of the Cellular Immunity of Newborn Harbor Seals (Phoca Vitulina). Archives of Environmental Contamination and Toxicology,55 (1), 129-136. doi: 10.1007/s00244-007-9092-3</ref>) found that lymphocytes of seal pups are particularly susceptible to the toxic effects of metals in the newborn period and that this susceptibility decreases subsequently.[[Image:Kakuschke_6.jpg|thumb|300px|right|'''Figure 5''': In cooperation with the FTZ Büsum seals were caught in the Danish and German Wadden Seas, specifically at the Islands Rømø and Helgoland and the sandbank Lorenzenplate. The seals were caught with a long net and for further investigations put in small individual nets. Several clinical parameters were collected and blood samples were taken. Additional blood samples were taken from pups during rehabilitation in the Seal Station Friedrichskoog.]]
 
  
===Stress – effects on the immune system===
+
=== High susceptibility of the immune system of pups to the toxic effect of metals===
The cytokine expression can be modulated by numerous factors, including stress. Fonfara et al. (2007<ref name="F">Fonfara S., Siebert, U. Prange, A. & Colijn, F. (2007). The impact of stress on cytokine and Haptoglobin mRNA expression in blood samples from harbour porpoises (Phoconea phocoena). Journal of the Marine Biological Association of the United Kingdom, 87, 305-311.
 
</ref>) compared cytokine mRNA expression from harbour porpoises exposed to different environments. Blood samples were taken from two healthy porpoises living in captivity at the Fjord and Belt Centre Kerteminde, Denmark, and from four wild porpoises accidentally caught in Danish waters. The results are suggestive of stress-induced modulation of the immune responses in the accidentally caught animals (Fig. 4, Fig. 5).
 
  
===Challenges===
+
Pups are exposed to metals through transplacental transfer, maternal milk and, at a later stage, contaminated prey. Kakuschke et al. (2008)<ref name="K5">Kakuschke, A., Valentine-Thon, E., Fonfara, S., Griesel, S., Siebert, U. & Prange, A. (2008). Metal-Induced Impairment of the Cellular Immunity of Newborn Harbor Seals (Phoca Vitulina). Archives of Environmental Contamination and Toxicology,55 (1), 129-136. doi: 10.1007/s00244-007-9092-3</ref> reported that lymphocytes of new born seal pups are particularly susceptible to the toxic effects of metals, which subsequently decreases with age (Fig. 3). Additionally, the chemical form of the metal compound was shown to play an important role for the immunological effect. Four different mercury compounds were reported to have a suppressive effect on lymphocyte proliferation; however, differences were present between juvenile and adult seals, and depending on the chemical form of mercury. (Kakuschke ''et al''. 2010<ref name="K6">Kakuschke, A., Valentine-Thon, E., Fonfara S., Kramer, K. & Prange, A. (2010). Influences of methyl-, phenyl-, ethylmercury and mercurychloride on immune functions in harbour seals. Journal of Environmental Sciences, 21, 1716–1721.</ref>.)
Anthropogenic influences may lead to changes of the health status of animals. A set of reliable health parameters enables us to investigate routinely a high number of animals and to obtain information from indicators of the coastal ecosytem health, monitored in support of the Trilateral Monitoring and Assessment Program.
+
[[Image:Kakuschke_6.jpg|thumb|300px|right|'''Figure 5''': In cooperation with the FTZ Büsum seals were caught in the Danish and German Wadden Seas, specifically at the Islands Rømø and Helgoland and the sandbank Lorenzenplate. The seals were caught with a long net and for further investigations put in small individual nets. Several clinical parameters were collected and blood samples were taken. Additional blood samples were taken from pups during rehabilitation in the Seal Station Friedrichskoog.]]
  
==See also==
+
==Stress – effects on the immune system==
===Internal links===
+
The cytokine expression can be modulated by numerous factors, including stress. Cytokine mRNA expression from two harbour porpoises living in captivity and four accidentally caught wild living porpoises were compared. A stress-induced modulation of the cytokine expression was suspected in the accidentally caught wild animals (Fig. 4, Fonfara ''et al''. 2007 <ref name="F">[http://www.vliz.be/imis/imis.php?module=ref&refid=119044 Fonfara S., Siebert, U. Prange, A. & Colijn, F. (2007). The impact of stress on cytokine and Haptoglobin mRNA expression in blood samples from harbour porpoises (Phoconea phocoena). Journal of the Marine Biological Association of the United Kingdom, 87, 305-311.]</ref> <ref name="F2">Fonfara S., Siebert, U., Prange, A (2007). Cytokines and acute phase proteins as markers for infection in harbour porpoises (Phoconea phocoena). Marine Mammal Science 23 (4): 931-942.</ref>).
 +
Furthermore, cytokine and acute phase proteins transcription varied in harbour seal pups during rehabilitation in the seal station Friedrichskoog, Germany, suggesting that these parameters might be useful to assess the health status, maturation of the immune system, and the ability to handle stress in these animals (Fig. 5, Fonfara ''et al''. 2008 <ref name="F5">Fonfara, S., Kakuschke, A., Rosenberger, T., Siebert, U. & Prange, A. (2008). Changes of cytokine and acute phase protein expression in blood samples of harbour seal pups during their first months of life. Marine Biology, 155, 337-345.</ref>).
 +
 
 +
==Acute phase proteins (APPs) – long term studies and protein characterization==
 +
 
 +
Protein blood concentrations of APPs such as haptoglobin (Hp), C-reactive protein (CRP), or transferrin are influenced by several diseases and might, therefore, be useful parameters to monitor health. Long term studies of Hp and CRP levels in harbour seals between 2002 and 2007 showed variations between years, as well as differences between age groups and sex (Kakuschke et al., 2010 <ref name="K7">Kakuschke, A., Erbsloeh, H.-B., Griesel, S. & Prange, A. (2010). Acute phase protein haptoglobin in blood plasma samples of harbour seals of the Wadden Sea and of the isle Helgoland. Comparative Biochemistry and Physiology, Part B, 155, 67–71.</ref>, 2012 <ref name="K8">Kakuschke, A.; Pröfrock, D.; Prange, A. (2013). C-reactive protein in blood plasma and serum samples of harbour seals (Phoca vitulina). Marine Mammal Science, doi:10.1111/j.1748-7692.2012.00603.x</ref>). Rosenfeld ''et al''. 2009 <ref name="R">Rosenfeld, H., Lassen, S. & Prange, A. (2009). Characterization of haptoglobin in the blood plasma of harbor seals (Phoca vitulina). Journal of Proteom Research, 8, 2923-2932.</ref>) analyzed the chemical structure and isoforms of Hp isolated from Wadden Sea harbor seals, Grebe ''et al''. analysed structure and isoforms of transferrin and obtained reference ranges for North Sea seals (Grebe ''et al''. 2010 <ref name="G1">Grebe, M., Pröfrock, D., Kakuschke, A., Broekaert, J.A.C. & Prange, A. (2010). Metallomics approach for the identification of the iron transport protein transferrin in blood from harbor seals (Phoca vitulina). Metallomics, 2, 661-720.</ref>, 2011 <Ref name="G2">Grebe, M., Pröfrock, D., Kakuschke, A., Broekaert, J.A.C. & Prange, A. (2011). Fast and reliable absolute quantification of transferrin in blood samples of harbour seals using HPLC-ICP-MS. Metallomics, 3, 176-185.</ref>, 2012 <ref name="G3">Grebe, M., Pröfrock, D., Kakuschke, A., del Castillo Busto, M.E., Montes-Bayon, M., Sanz-Medel, A., Broekaert, J.A.C. & Prange, A. (2012). Comparison of different methods for the absolute quantification of harbour seal transferrin glycoforms using HPLC-ICP-MS. Journal of Analytical Atomic Spectrometry, 27, 440-448.</ref>).
 +
 
 +
==Challenges==
 +
Anthropogenic influences may have an effect on the health status of animals. Reliable health parameters allow routine investigations of a large number of animals and provide information about these animals used as indicators of the coastal ecosystem, monitored in support of the Trilateral Monitoring and Assessment Program.
 +
 
 +
==Related articles==
 +
* [[Common biomarkers for the assessment of marine pollution]]
 +
* [[Endocrine system]]
 
* [[Elemental mass spectrometry - a tool for monitoring trace element contaminants in the marine environment]]
 
* [[Elemental mass spectrometry - a tool for monitoring trace element contaminants in the marine environment]]
 
* [[Acoustic monitoring of marine mammals]]
 
* [[Acoustic monitoring of marine mammals]]
* [[Common biomarkers for the assessment of marine pollution]]
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* [[Coastal pollution and impacts]]
 +
* [[Environmental risk assessment of marine activities]]
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* [[Endocrine disrupting compounds in the coastal environment]]
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* [[Portal:Ecotox]]
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* [[Threats to Coral Reefs: the Effects of Chemical Pollution]]
  
 
==References==
 
==References==
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[[Category:Marine habitats and ecosystems]]
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[[Category:Ecotoxicology]]  
 
[[Category:Coastal and marine pollution]]
 
[[Category:Coastal and marine pollution]]
[[Category:Biological processes and organisms]]
 

Latest revision as of 14:12, 6 September 2020


Introduction

Figure 1: The health of the Harbour Seal as top predator is an important biological parameter of the Trilateral Monitoring and Assessment Program (TMAP). This program was founded by The Netherlands, Denmark and Germany, for the protection and conservation of the Wadden Sea. It includes management, monitoring and research, as well as political matters.
Figure 2:Overview of immunological investigations using blood samples of marine mammals.

Marine mammals are top predators of the marine food web and can therefore be used as indicators for the ecosystem (Trilateral Monitoring and Assessment Program, TMAP, Fig. 1). Industrial human activities, e.g. fisheries, offshore drilling and wind power generation, as well as pollutants in the environment affect the North and Baltic Sea ecosystems, which are the habitat of marine mammals such as harbour porpoises (Phocoena phocoena), harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus). The present article reports tools to investigate the health status of harbour seals. A dysregulation of the immune system can lead either to suppression or an increased sensitivity (hypersensitivity) of the immune system, which might affect morbidity and mortality in these animals. Parameters of the immune system were investigated as stable biomarkers for disease processes. Furthermore, factors indicating the impact of pollutants were identified and chemically characterised.

Methodology

The following parameters of the immune system can be measured in blood samples of captive and free-ranging animals using in-vivo measurements, in-vitro cell culture experiments, and bioanalytical methods (Fig. 2): lymphocyte proliferation, mRNA and protein expression of cytokines and acute phase proteins (APP). Protein structures, and the influence of metal and organic pollutants on these can be analysed and allow, therefore, conclusions about the relevance of the pollutant. Elements indicating the relationship between pollutant burden and health parameters can be analysed in body fluids using mass spectrometry (see article Elemental mass spectrometry).

Figure 3: Directly after arrival in the Seal Station the lymphocytes of newborns were particularly susceptible to the toxic effect of metals. A lot of metals tested e.g. beryllium, lead and cadmium inhibit the lymphocyte proliferation (value <0.1). This effect decreased during the time of rehabilitation.

Measurement of lymphocyte proliferation

Lymphocytes are isolated from blood samples and cultured with and without ions of metal pollutants using a lymphocyte transformation test (LTT, Fig. 2). After an incubation period, transformation and proliferation of the lymphocytes is examined and a stimulation index calculated. Depending on degree of stimulation the reactivity of the lymphocytes and therefore immune system can be detected.

Quantification of cytokine expression

mRNA and protein expression of cytokines and APP can be measured by real time reverse transcriptase polymerase chain reaction (RT-PCR) and mass spectrometry, respectively. Next to quantification of protein concentrations, mass spectrometry allows also the characterization of protein isoforms (Fig. 2). Cytokines as interleukin-1, -2, -4, -6, -10, -12, tumour necrosis factor-alpha, transforming growth factor-beta and APP (e.g. haptoglobin, C-reactive protein, transferrin, metallothionein, heat shock protein) are stimulated by infection, inflammation, trauma or intoxication. They might therefore be helpful to identify an activated immune system and to monitor the health status.

Figure 4: Cytokine index of IL-2 mRNA from the blood samples of two harbour porpoises living in captivity (Pp1, Pp2) and four accidentally caught animals (Pp3-Pp6)(Fonfara et al. 2007)

Metal pollution – effects on the immune system

In many areas of the industrialised world pollution by metals is present, which might have an effect on the immune competence of free-ranging marine mammal populations. An imbalance of the immune system caused by pollutants has been suggested to play a role in the incidence of infectious diseases in marine mammals (Jepson et al., 1999[1]; Bennett et al., 2001[2]). Metals affect the function of immune cells by a variety of mechanisms. Depending on the particular metal, its speciation, concentration and bioavailability, permanent metal exposure can result in suppression or stimulation of the immune system.

Metal induced hypersensitivity in seals

A chronic exposure to metal pollutants and the intake via food and water was suspected to cause hypersensitivity reactions in marine mammals. Hypersensitivity reactions were found for Mo > Ni >Ti > Cr, Al > Pb, Be, Sn, (in decreasing frequency as indicated by the present order) in several pinnipeds from the North Sea (Kakuschke, 2006[3]) and a relationship between blood concentrations and hypersensitivity reactions of the particular metals was present (Kakuschke et al., 2005 [4]). Furthermore, an association between lymphocyte proliferation caused by Ni and Be and cytokine expression was detected in a study of a grey seal (Kakuschke et al., 2006[5]). Changes in haematological parameters and liver enzymes in seals with a metal-induced hypersensitivity suggests that hypersensitivity reactions are a complex phenomenon (Kakuschke et al., 2011 [6]). These findings indicate a chronic exposure of marine mammals of the North Sea to metal pollutants.


High susceptibility of the immune system of pups to the toxic effect of metals

Pups are exposed to metals through transplacental transfer, maternal milk and, at a later stage, contaminated prey. Kakuschke et al. (2008)[7] reported that lymphocytes of new born seal pups are particularly susceptible to the toxic effects of metals, which subsequently decreases with age (Fig. 3). Additionally, the chemical form of the metal compound was shown to play an important role for the immunological effect. Four different mercury compounds were reported to have a suppressive effect on lymphocyte proliferation; however, differences were present between juvenile and adult seals, and depending on the chemical form of mercury. (Kakuschke et al. 2010[8].)

Figure 5: In cooperation with the FTZ Büsum seals were caught in the Danish and German Wadden Seas, specifically at the Islands Rømø and Helgoland and the sandbank Lorenzenplate. The seals were caught with a long net and for further investigations put in small individual nets. Several clinical parameters were collected and blood samples were taken. Additional blood samples were taken from pups during rehabilitation in the Seal Station Friedrichskoog.

Stress – effects on the immune system

The cytokine expression can be modulated by numerous factors, including stress. Cytokine mRNA expression from two harbour porpoises living in captivity and four accidentally caught wild living porpoises were compared. A stress-induced modulation of the cytokine expression was suspected in the accidentally caught wild animals (Fig. 4, Fonfara et al. 2007 [9] [10]). Furthermore, cytokine and acute phase proteins transcription varied in harbour seal pups during rehabilitation in the seal station Friedrichskoog, Germany, suggesting that these parameters might be useful to assess the health status, maturation of the immune system, and the ability to handle stress in these animals (Fig. 5, Fonfara et al. 2008 [11]).

Acute phase proteins (APPs) – long term studies and protein characterization

Protein blood concentrations of APPs such as haptoglobin (Hp), C-reactive protein (CRP), or transferrin are influenced by several diseases and might, therefore, be useful parameters to monitor health. Long term studies of Hp and CRP levels in harbour seals between 2002 and 2007 showed variations between years, as well as differences between age groups and sex (Kakuschke et al., 2010 [12], 2012 [13]). Rosenfeld et al. 2009 [14]) analyzed the chemical structure and isoforms of Hp isolated from Wadden Sea harbor seals, Grebe et al. analysed structure and isoforms of transferrin and obtained reference ranges for North Sea seals (Grebe et al. 2010 [15], 2011 [16], 2012 [17]).

Challenges

Anthropogenic influences may have an effect on the health status of animals. Reliable health parameters allow routine investigations of a large number of animals and provide information about these animals used as indicators of the coastal ecosystem, monitored in support of the Trilateral Monitoring and Assessment Program.

Related articles

References

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The main authors of this article are Kakuschke, Antje, Kramer, Katharina and Fonfara, Sonja
Please note that others may also have edited the contents of this article.

Citation: Kakuschke, Antje; Kramer, Katharina; Fonfara, Sonja ; (2020): Marine mammals' health as an indicator of ecosystem health - tools for monitoring. Available from http://www.coastalwiki.org/wiki/Marine_mammals%27_health_as_an_indicator_of_ecosystem_health_-_tools_for_monitoring [accessed on 28-03-2024]